The jelllyfish Aequorea forskalea emits bright green bioluminescence when stimulated mechanically, electrically or chemically. Two proteins are primarily responsible for the light. One is aequorin, the actual source of the bioluminescence (whose emission maximum is at 469 nm) and the other is a green fluorescent protein (GFP) which accepts energy from aequorin (in vivo) and emits green light (Lambda max 510 nm). The sole requirement for aequorin bioluminescence is the presence of Ca++ (or a variety of other divalent and trivalent metal ions including the lanthanides). A substrate (luciferin) and oxygen are trapped within the protein; the binding of Ca++ to the protein inspires a rapid catalytic oxidation of the luciferin by some form of oxygen which results in emission of light. After being thus discharged, the protein acquires a bright blue fluorescence. Aequorin turns over only once in response to binding Ca++. The green fluorescent protein structurally extremely stable and the fluorophore is an intrinsic part of the protein is backbone presumably arising by a unique post-translational modification. For the next grant period we propose to determine how the protein binds oxygen and to investigate the intramolecular mechanisms of oxidation of the luciferin. We will then do a detailed study of the photophysics of the fluorescence processes of both aequorin and GFP and investigate the mechanisms underlying bioluminescence energy transfer. We will use fluorescence spectroscopic methods to probe the dipolar character of the environment of the fluorophore in both proteins and to determine the dynamics of the fluorophores through measurements of time resolved anisotropy in the picosecond domain by use of multiple frequency phase fluorometry. Luminescence lifetime and circular polarized luminescence measurements of Tb(III) and Eu(III) bound to aequorin will be used to determine the coordination chemistry at the lanthanide binding sites. We also propose to determine the aminoacid sequence of GFP and to investigate the biochemical mechanisms mediating the post-translational synthesis of the green fluorescent moiety.